Adaptive response of Yersinia pestis to extracellular effectors of innate immunity during bubonic plague Florent Sebbane*†, Nadine Lemaıˆtre*‡§, Daniel E. Sturdevant¶, Roberto Rebeil*ʈ, Kimmo Virtaneva¶, Stephen F. Porcella¶, and B. Joseph Hinnebusch*,** *Laboratory of Zoonotic Pathogens and ¶Genomics Core Facility, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; ‡Institut National de la Sante´et de la Recherche Me´dicale Unite´801 and Faculte´deMe´ decine Henri Warembourg, Universite´de Lille II, Lille F-59045, France; and §Institut Pasteur, Lille F-59021, France Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved June 13, 2006 (received for review February 11, 2006) Yersinia pestis causes bubonic plague, characterized by an en- phagocytic oxidative burst that generates antimicrobial reactive larged, painful lymph node, termed a bubo, that develops after oxygen species (ROS), down-regulate the normal proinflamma- bacterial dissemination from a fleabite site. In susceptible animals, tory response, and induce apoptosis of dendritic cells, NK cells, the bacteria rapidly escape containment in the lymph node, spread macrophages, and polymorphonuclear neutrophils (PMNs) (3, 4, systemically through the blood, and produce fatal sepsis. The 6–8). Although substantial, most of the experimental evidence fulminant progression of disease has been largely ascribed to the for these models comes from in vitro studies with the less virulent ability of Y. pestis to avoid phagocytosis and exposure to antimi- enteric pathogens Yersinia pseudotuberculosis and Yersinia en- crobial effectors of innate immunity. In vivo microarray analysis of terocolitica or from studies using attenuated strains of Y. pestis Y. pestis gene expression, however, revealed an adaptive response injected directly into the blood stream. to nitric oxide (NO)-derived reactive nitrogen species and to iron Bubonic plague does not invariably progress to septicemic limitation in the extracellular environment of the bubo. Polymor- plague, even in susceptible animals, indicating that the lymph phonuclear neutrophils recruited to the infected lymph node ex- node is the deciding arena of immune response success or failure. pressed abundant inducible NO synthase, and several Y. pestis To better understand host–pathogen interactions in this key homologs of genes involved in the protective response to reactive arena, we investigated Y. pestis adaptation to the innate immune nitrogen species were up-regulated in the bubo. Mutation of one response and other host factors in the bubo by in vivo gene of these genes, which encodes the Hmp flavohemoglobin that expression profiling and virulence testing of select mutants using detoxifies NO, attenuated virulence. Thus, the ability of Y. pestis to a previously characterized rat model of bubonic plague (2). destroy immune cells and remain extracellular in the bubo appears to limit exposure to some but not all innate immune effectors. High Results NO levels induced during plague may also influence the developing Transcriptional Profile of Y. pestis in the Bubo. Within a few days adaptive immune response and contribute to septic shock. after injecting Y. pestis into the dermis of the upper pelvic region, rats develop classic signs of bubonic plague, including a grossly inducible nitric oxide synthase ͉ reactive nitrogen species enlarged inguinal lymph node and limping and reluctance to move the associated hind leg. The primary bubo in rats with 7 9 Y. pestis ersinia pestis, the agent of plague, primarily affects rodents terminal plague contains 10 to 10 (2), and sufficient bacterial RNA was recovered from the contents of a single but is also one of the most invasive and virulent bacterial Y inguinal bubo for microarray analysis (Fig. 5, which is published pathogens of humans (1). Bubonic plague, the most common as supporting information on the PNAS web site). To identify form of the disease in rodents and humans, is usually acquired bacterial genes that are differentially expressed during disease, from the bite of an infected flea. Disease progression and we compared the transcriptional profiles of Y. pestis isolated histopathology of plague in the rat closely resemble those of from buboes and from laboratory cultures. The gene expression human bubonic plague (2). From an intradermal inoculation site, patterns were reproducible and discrete for each of the different Y. pestis rapidly disseminates through afferent lymphatic vessels in vivo and in vitro conditions, with the bubonic profile most to the regional draining lymph nodes. After entering the sub- similar to that of stationary-phase bacteria cultured at 37°C capsular sinus of the node, the bacteria rapidly multiply and (Fig. 1). spread throughout the entire parenchyma of the node, resulting Among the Y. pestis genes most highly expressed in the bubo in lymphadenitis characterized by a painful, swollen lymph node were those for known virulence factors (1), such as the antiph- termed a bubo. Histologically, primary buboes of rats and agocytic F1 protein capsule, a plasminogen activator required for humans contain extensive masses of extracellular Y. pestis, systemic invasion from the lymph node, the iron acquisition necrotic lymphoid tissue, hemorrhage and fibrin, and are sur- system encoded on the Y. pestis pathogenicity island, and the rounded by an edematous perinodal capsule (2). Without early antibiotic treatment, bubonic plague usually progresses rapidly to septicemic plague, a form of the disease characterized by Conflict of interest statement: No conflicts declared. bacteremia, systemic spread, and life-threatening Gram-negative This paper was submitted directly (Track II) to the PNAS office. sepsis. Hematogenous spread to the lungs can also result in Freely available online through the PNAS open access option. pneumonic plague. Abbreviations: RNS, reactive nitrogen species; ROS, reactive oxygen species; iNOS, inducible Current models of plague pathogenesis emphasize the impor- nitric oxide synthase; TTSS, Type III secretion system; PMN, polymorphonuclear neutrophil; tance of the bacterial Type III secretion system (TTSS) in GSNO, nitrosoglutathione. evading innate immunity, the first line of defense against infec- Data deposition: The MIAME (Minimum Information About a Microarray Experiment) data tion (1, 3, 4). The Y. pestis TTSS consists of at least 42 genes have been submitted to the Gene Expression Omnibus repository (accession no. GSE3793). arranged in several operons on a 70.5-kb plasmid that is essential †Present address: Institut National de la Sante´ et de la Recherche Me´dicale Unite´ 801, for virulence (5). These genes encode virulence proteins, termed Universite de Lille II, and Institut Pasteur, Lille F-59021, France. Yops, and a delivery system to inject the Yops into eukaryotic ʈPresent address: Sandia National Laboratories, Albuquerque, NM 87185. cells (3). The translocated Yops disrupt a variety of eukaryotic **To whom correspondence should be addressed. E-mail: [email protected]. cell signaling pathways to inhibit phagocytosis, attenuate the © 2006 by The National Academy of Sciences of the USA 11766–11771 ͉ PNAS ͉ August 1, 2006 ͉ vol. 103 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601182103 Downloaded by guest on September 25, 2021 flavoglobin that is induced by NO and confers resistance to RNS in vitro and in macrophages (12–15), increased 10- to 20-fold in the bubo. In addition, metL, which is required for synthesis of the NO antagonist homocysteine and for Salmonella resistance to GSNO (16), and ytfE (dnrN), which is required for Escherichia coli resistance to NO (14), were both up-regulated 3- to 12-fold. Up-regulation of hmp, ytfe (dnrN), and other genes that may be involved in protection against RNS, such as hcp, hcr, and tehB, has recently been attributed to repression of the NO-sensitive negative regulator encoded by yjeB (nsrR) (17, 18). Expression of the Y. pestis nsrR homolog (YPO0379) was down-regulated 1.7- to 4-fold in the bubo compared with stationary-phase, in vitro cultures, and hcr, tehB, hmp, and ytfE (dnrN) were up-regulated (Fig. 2B). Y. pestis homologs of other genes, which have been consistently found to be induced in E. coli by in vitro exposure to RNS (13–15), such as the nrdHIEF ribonucleotide reductase operon, were also up-regulated in the bubo (Fig. 2B). Fig. 1. Principal component analysis of microarray data from six biological replicate samples for each of the in vitro and in vivo conditions. The first two Localized and Systemic Induction of Inducible NO Synthase (iNOS) principal components show the separation of the bubo-specific expression During Bubonic Plague. NO-derived antimicrobial activity is gen- profile from the expression profiles in exponential and stationary-phase erated by macrophages, PMNs, and other host cells by the iNOS cultures grown at 21°C and 37°C. Ellipses encompass exponential, stationary, and rat samples. in response to infection and is an important component of the innate immune response to intracellular pathogens (19, 20). Both PMNs and macrophages are recruited to the rat bubo (2), and TTSS (Table 1, which is published as supporting information on we detected large numbers of iNOS-expressing cells, which the PNAS web site). The histopathology and transcriptome of Y. appeared to be primarily PMNs, in close association with pestis in the bubo are consistent with the predicted antiphago- extracellular